TWI290226B - Method for detecting human HCCR-1 protein and a kit for diagnosing liver cancer using the same - Google Patents

Abstract

Provided are a method for detecting liver cancer by measuring the level of expression of HCCR-1 protein in a specimen sample in vivo by an antigen-antibody binding reaction using an antibody specifically binding to a protein expressed from human proto-oncogene HCCR-1, and a kit for diagnosing liver cancer using the same. The diagnosing method and kit can be advantageously used for early diagnosis of liver cancer and for diagnosis of small HCC due to improved accuracy and reproducibility compared to the conventional AFP (alpha-fetoprotein) test.

Description

1290226 玖Invention Description: [Technical Field] The present invention relates to an antigen-antibody binding reaction for detecting a sample by using an antibody that specifically binds to a protein expressed from a human proto-oncogene A method comprising a human HCCR-1 protein, and a kit for use in diagnosing liver cancer. [Prior Art] In general, in order to increase the survival rate of patients with liver cancer, the most important one is to detect Hcc (hepatocellular carcinoma) in the early stage by letting people with high risk of liver cancer go to the hospital for regular health tests. The presence. A person with a high risk of developing liver cancer is called a high-hepatic cancer risk group. Most of the risk factors associated with liver cancer are classified into physiological factors and pathological factors. Examples of physiological factors include age, gender, ethnicity, etc., and examples of pathological factors include hepatitis B virus or hepatitis C virus infection, cirrhosis, alcohol abuse, exposure to aflatoxin, and relatively rare metabolism. Diseases, sex hormones, chemicals, etc. So far, according to the fact that the concentration of AFP in normal adult serum is almost zero, but it is found in many patients with liver cancer, the sputum is increased; the fetal protein level is favorably used in the diagnosis of early detection of liver cancer. One of the screening factors. However, in fact, the concentration of MP will not only increase in liver cancer but also in benign diseases such as chronic hepatitis or cirrhosis, malignant diseases such as choriocarcinoma or hepatoblastoma, or pregnancy, etc. Increased among them. Conversely, it has been reported that AFP cannot be advantageously used for the early diagnosis of liver cancer, as indicated by many clinical trials that indicate that AFP concentrations in early hcc sera are substantially normal (Collier J and Sherman M? Hepatology 27:273 -278, 1998; Okuda K5 J. Hepatol 32 (1 Suppl): 225-237, 2000). According to this report, there was a non-specific increase in serum fine levels observed in some patients with chronic hepatitis or cirrhosis but 1290226 without HCC. Moreover, approximately HCCs patients exhibit normal AFP levels, and more than 8% of patients with small HCCs with a diameter of no more than 2 cm show no statistical difference in AFP levels, making fresh and normal adults substantially Can't compare. Regarding whether the HCC patient's body _ AFP level is suitable for the secret, there are several previous reports that can be mentioned as τ. There are - purely disambiguated (four) 46.2% m patients with a direct financial situation of 5 _ hccs _ AFP levels are substantially normal. Another report showed that about 25.5% of patients with HCCs with a diameter of no more than 5 cm had an App level that was normal and about 34.8% of patients with a relatively small diameter HCC of no more than 3 cm. The normal AFP levels are exhibited (Collier J and Sherman M, // epiato/og); 27: 273_278, 1998; κ, 』 /fepato/· 32 (1 Suppl): 225-237, 2000). For this reason, although the Αρρ test is considered to be a necessary step to select a patient for HCCs in patients with chronic liver disease to some extent, the diagnosis of liver cancer based on AFP levels requires many considerations. Therefore, there is a need to develop a new serological test that has far greater sensitivity and specificity than traditional AFP testing. Some of these studies have recently found that the lectin_reactive fracti〇ns will vary depending on the various subtypes of the AFP diol series structure (giycol_series structures). According to the findings of the subtypes of AFP-L3 that are more specific for liver cancer, a report mentions that a more specific diagnostic test for liver cancer can be made by measuring the AFP-L3 part of the App test (Okuno M et al ·, 』G as a coffee she ra /. / ^ 7 steal /. 16 (12): 1329_1335, 2001). However, due to several limitations associated with clinical diagnosis, such an attempt cannot be used as a useful tool for the use of a liver cancer rupture tool. 1290226 Another possible diagnostic test is ultrasound screening, which can be implemented in a simple, safe manner. However, this method has poor predictive ability and cannot detect small lesions smaller than 丨 cm in diameter. Moreover, since this technique relies on the inspector's skill or experience, it may lead to quite different results based on the inspector in terms of diagnostic accuracy, and it can be inferred that it lacks objectivity. Furthermore, it is quite difficult to apply ultrasound to patients suffering from severe cirrhosis and having a high risk of liver cancer, so that it is substantially incapable of accurately identifying the lesion. Therefore, in order to achieve early detection of cancer, it is highly desirable to develop a new serological test with improved sensitivity and specificity. In summary, in order to develop an effective test method for early diagnosis of liver cancer, the inventors of the present invention have carefully studied and identified the use of proteins that specifically bind to human protooncogene HCCR-1. The antigen-antibody-binding reaction of the antibody can effectively detect the liver cancer, and the human pro-oncogene HCCm is deposited in the same name as that described in Korean Patent Publication No. 2-39556. This finding led the inventor of the present invention to complete the present invention. SUMMARY OF THE INVENTION In order to solve the above-mentioned problem, the present invention proposes a method for effectively detecting an occupational oncogene HCCR 1 at an early stage and using a kit for diagnosing liver cancer. For the purpose of the above-mentioned present, it is proposed to measure a sample in vivo by using an anti-shipping antigen-antibody binding reaction which specifically binds to the human pro-oncogene HCCR_1 (4) protein f (in The method of diagnosing liver cancer is called the method of diagnosing liver cancer, and a set of 1290226 used for diagnosing liver cancer. [Simple diagram of the diagram] Figure 1 illustrates the sage of xian xian from lion to ρΜΑ£·ρ; 2χ/Ηα^ Escherichia coli (E. coli) BL21 strain isolated and purified HCCR_i recombinant protein to perform the results of immunospotting; Figure 2 illustrates the production of HCCR] recombinant protein as an antigen according to the present invention Screening results for fusion cell lines that produce HCCR-1 early strain antibodies; Figure 3 illustrates the results of immunoassays for Hcc tissues using a sputum monoclonal antibody in accordance with the present invention, wherein A and B are shown as HCC The results of the immunoassay of the tissue, c shows the results of the immunoassay of the hepatitis tissue, and D shows the results of the immunoassay of the normal liver tissue; and Figures 4a to 4e illustrate the use of the HCCR_i single according to the present invention. The diagnostic kit of antibodies detects the serum of HCC and hepatitis patients, the results of liver cancer diagnosis of hccrj protein in maternal serum and normal serum, wherein Figure 4a shows the absorbance of serum from HCC patients, and Figure 4b shows the results from hepatitis. The absorbance of the patient's serum, Figure 4c shows the absorbance of the maternal serum' Figure 4c shows the absorbance of normal serum, and Figure 4e shows the results of the comparison of the absorbance levels of the individual samples from the cutoff value of the standard curve. Hereinafter, specific examples of the present invention will be described in detail with reference to the accompanying drawings. Human proto-oncogene HCCR-1 (GenBank Accession No. AF195651; Korean Patent Publication No. 2001-39556), which is located on chromosome 12 ( The long arm of Chromosome No. 12 has an open editing frame (0Penreading frame), which has the function of becoming an oncogene when it is overexpressed in the mammalian body, and is derived from the gene. A protein having a size of about 4〇kDa (referred to as HCCR) protein. HCCR_1 protein-specific antibody is preferred The antiserum obtained by immunizing the antigenic protein in the animal is purified. More preferably, the Ηα: ΙΜ antigen protein-specific antibody is purified from the serum or egg known by immunizing HCCR-1 protein in the hen. The antibody may be a plurality of antibodies or a monoclonal antibody. To synthesize an antibody of HCOM protein f, we need to obtain hccim protein. HCCR-1 protein can be synthesized by genetic acid engineering or genetic engineering. The law is made into a new egg. For example, Sha can be prepared by the following method: using the hccr] gene sequence listed in the NIH program GenBank database to prepare a recombinant protein expression vector of the egg (4) The human proto-oncogene HCCR-1 recombinant protein is isolated/purified by transforming the expression vector into E. coli to obtain a transformant for producing the HCCR 1 recombinant protein; and culturing the transformant.

In a preferred embodiment of the present invention, a recombinant protein having a maltose having a face-to-team end, which is transformed into a large intestine rod _blu containing pmcp2X/HCCR_l containing a hccr heart gene, is then isolated by an appropriate enzyme/ Purification to transfer both the CR_1 protein of maltose as an antigenic protein. In order to identify the protein thus produced as a species of HCCIU heavy, and the protein was observed by Western blot analysis, the specificity of the HCCR-1 recombinant protein having a molecular weight of about % kDa was observed. The fusion tumor cell 1290226 line which produces monoclonal antibodies by immunization and cell fusion of mice by screening and analyzing from the scales of the large scales and purifying (4) eggs is used as an antigen. The immunized mouse line required to develop a fusion tumor cell line is thoroughly mixed with a considerable amount of Freimd, S complete adjuvant until the mixture is emulsified, and then intraperitoneally injected into the mouse, followed by intraperitoneal injection into the mouse. Intensive injections are produced to increase the immunogenicity of mice. Preferably, subsequent booster injections are administered to the mice three times via the intraperitoneal route using only HCCR-1 recombinant protein without the use of an adjuvant. Mouse sera immunized by injection of HCCR-1 recombinant protein antigen were collected and antibody titers were measured. The spleen cells of the antibody-forming mouse spleen cells were mixed with the cells in a ratio of the number of cells of 〇: 且 and then fused in polyethylene glycol, and then the medium was added for dilution. The fused tumor cells are then transferred to a screening medium and cultured immediately. At the time when the growth of the fusion tumor cell group was clearly observed, the medium was changed to a selection medium to promote cell proliferation. Useful cells Examples of the fusion mother cells include the SP2/0 Agl4 cell line, the P3x653 cell line, and the NS-1 cell line. Examples of the medium which can be used include PMI medium, dmem medium, imdm medium, and F12 medium. Examples of useful screening media include jjat media. In the fusion tumor cell group grown in the screening medium, only the fusion tumor cell group capable of specifically reacting with the HCCR-1 protein antigen was selected by the mdifeet enzyme-linked immunosorbant assay (ELISA). do not. In other words, the absorbance is measured with an eusa reader to screen for a fusion tumor cell line that secretes antibodies that are highly binding to the purified HCCR4 protein, and the fusion tumor cells are diluted by limiting dilution to make the screened cells single. A strain whereby a cell line composed of cells proliferating from a single cell is established. 1290226 The individual cell line of the human calamity gene HCCW protein is used to produce a large number of cell lines through the following steps: 1) Inject the fusion cell line into the small gas via the intraperitoneal route; 2) From small Rat's hypertrophied abdomen collects ascites fluid; and 3) rides from ascites fluid (IV) HCQM egg self-quality tree-specific antibody protein. The diagnosis of cancer by using the HCOM-specific anti-white (four) antigen-antibody binding reaction thus produced can be accomplished by measuring the in vivo expression of the protein in a sample taken from a sample. Performance levels can be obtained using techniques known in the art, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA), sandwich assays, and Western points on polypropylene gels. Analysis of the ulceration or immunization bl〇tting. In a preferred embodiment, the ELISA technique is performed using the Ηα:ΙΜ protein-specific antibody by the following steps: 1) The sample and the control sample are Placed in a reactor coated with hydrazine (:αΜ specific antibody to induce antigen-antibody reaction; 2) using secondary antibody-labeled complex (sec〇ndary antib〇dy撼d(10)_ Qualitative solution detection antigen-antibody reaction product; and 3) comparison of the detection results of the sample with the results of the control group. The present invention also proposes a protein wafer that can protect the HCCR-1 specificity by immobilizing the HCCW-specific antibody protein on the biomicrochip to facilitate the reaction between the sample and the in vivo sample collected from one body. The antigen of the antibody protein. A large number of samples can be analyzed using known techniques, such as eLisa, biomicrochips or automated microarray systems. Further, the present invention provides a kit for diagnosing liver cancer comprising an antibody reactive with HCCR4 specifically 11 1290226, thereby facilitating early diagnosis of liver cancer. The diagnostic kit of the present invention comprises: 1) a reactor coated with an HCCR-1 specific antibody; 2) a positive control group containing a HCCR-1 standard antigen and an animal containing the antigen injected therein a negative control group of antiserum; 3) a tortoise-like antibody composition having a flattened labeling substance which can react with a substrate to produce a color; 4) a color developing substrate solution, The labeling substance reacts to produce a color; 5) - for the washing solution in each step; and 6) an enzyme to stop the solution. The W-cut kit can diagnose liver cancer by quantitatively or qualitatively analyzing the antigen of the antibody protein via an antigen-antibody binding reaction. The antigen-antibody binding reaction can be detected by techniques commonly known in the art, including ELISA, RIA, sandwich assay, Western Point/Bei on polyacrylamide gel, and immunospot analysis. For example, the diagnostic kit can be provided for use in an ELISA using a 96-well microtiter plate coated with recombinant monoclonal antibody protein. Examples of the reactor for coating the antibody protein thereon include a nitrocellulose membrane, a 96-hole plate made of a polyethylene resin, a 96-hole plate made of a polystyrene resin, and a carrier glass. As described above, the antibody according to the present invention is preferably purified from an antiserum obtained by immunizing an antigenic protein in an animal. The HCCRq protein-specific antibody is preferably applied to the parent reactor at a ratio of about 1 to 1 μg/lQ〇 microliter. The control group contained in the diagnostic kit according to the present invention includes a positive control group and a negative control group. Yang Μ 10, the exhibition group is a mixture containing the standard antigen of HCCR-1 protein, and the negative control group is the serum of the animal which does not have 12 1290226 infected with HCCR-1 protein antigen. In the examples of the present invention, a plurality of protein concentrations are used: 0 ng/ml (ng/ml) (A), 2 ng/g (B), 4 ng/g (C), 80 m. Micrograms/ml (D), 160 ng/ml (E), 320 ng/ml (F) and 640 ng/ml (G) of HCCR_1 protein standard antigen solution. The secondary antibody-labeling substance is preferably a color developing agent using known inducible color development, and examples of the labeling substance usable in the present invention include horseradish peroxidase (HRp), alkaline phosphatase. , colloidal gold, luciferin such as poly-L-lysine _ fluorescein isothiocyanate (p〇ly L_lysine_fluorescein iS0thi0cyanate) (FITC) or rhodamine isothiocyanate (rh〇damine-B_is〇 Thi〇Cyanate) (RITC), and dyes. In the present invention, for example, a goat anti-rabbit IgG-HRP complex (g0at anti_rabbit c〇njugate) (IgG HRp composition) is used. Preferably, the color appearance used varies depending on the labeling substance included in the color development, and useful examples thereof include 3,3,5,5,4-tetramethylbenzidine (3,3,, 5,5, 担_邮^说(TMB) ' 2,2'-well base-bis(3_ethylbenzothiazoline-6-sulfonic acid) (2,2 ·&ζίηο-1^( 3_6%^ηζ(Λΐώζο1ή^_6-3ΐι1ίόιήο acid) (ABTS), with o-phenylenediamine (0PD). More preferably, the color appears to be supplied by the matrix to dissolve in a buffer solution (in the state of 〇丨Μ). This color develops a substance such as TMB which is decomposed by the HRP which is used as a labeling substance of the secondary antibody composition to produce a color-developing precipitate. The color level of the precipitate is observed by the naked eye, thereby measuring the HCCR4 protein. The presence of the antigen. The washing solution preferably comprises phosphate buffer, Naa, and Tween 2, more preferably 〇·〇2 Μ phosphate buffer, 〇·13 M NaC1, and 〇〇5% Tween 2〇 buffer solution. After the antigen-antibody binding reaction, after reacting with the secondary antibody on the antigen-antibody complex, an appropriate amount of 13 1290226 will be The raffinate is supplied to the reaction H. The washing with the silk solution is repeated 3 to 6 times. Preferably, a phosphate buffer containing G_1% BSA is used as a blocking solution and preferably 2N is used. The sulfuric acid solution is used as an enzyme reaction stop solution. At this point, the antigen in the female product is used to detect the antigen in the female product at an early stage. The HOW single Wei body is divided into the heterozygous and negative control groups. The reaction of the impurity is detected, and the silk solution is added. In the obtained recording towel, the secondary residue which is marked with the label f which can be colored with the reverse stitching color is added, and the washing solution is used for washing. Adding a solution containing f to the obtained product to induce color development. Then 'measuring the absorbance at 4〇5 nm. Here, the average absorbance of the standard antigen solution A is greater than or equal to 0·_ to the scale Yu Gang. The average absorbance of the standard anti-county liquid f is greater than or equal to 1.200 and less than or equal to 3._. The average value between the absorbances of the standard antigen solutions A and f is - the rest is like the filament (4) is miscellaneous Reduced or negative sample. When the test _ sucking money is greater than the absorption of fresh anti-filament F, _ Body dilution and re-measurement of its absorbance. The detection with high domain stop_absorbance is identified as positive, while the sample with absorbance below the cutoff value is identified as negative. The determined person is based on this person. Because Ηα:] Μ特纽抗 _ liver cancer diagnosis kit, for the test (four) of the blood phase of the liberation of the guardian, with a higher accuracy and reproducibility of the test tool. The monthly diagnostic kit can be used for even the AFP-negative specimens. In summary, the diagnosis of liver cancer according to the present invention is __(10) and the diagnostic accuracy of 脑(10) is statistically effective. Sexually higher than the traditional AFP test. Moreover, even for the liver cancer tissues with negative results, a detection rate of about 93.1% _ is relatively high. Furthermore, the HCCR_1 diagnostic according to the present invention has a relatively high diagnostic performance of about 91.7% for small Hcc specimens having a diameter of no more than 2 cm than conventional App diagnostics. That is, when the normal liver tissue, the hardened tissue and the HCC tissue are stained by immunostaining using the HCCR_1 according to the present invention, no antigen which reacts with HCCR4 is detected in the normal liver tissue and the cirrhotic tissue, and Antigens that reacted with HCCR-1 only in HCC tissues exhibited a dark brown appearance. Therefore, the diagnostic method and the kit according to the present invention can be very advantageously used for the early diagnosis of liver cancer and the diagnosis of small HCC, because they have high accuracy and reproducibility relative to the conventional App test, which is to be implemented by the following The invention is illustrated in detail, but is not intended to limit the scope of the invention to the particular embodiments described. <Example 1> Production of HCCR_1 polyclonal antibody<1-1> Production of HCCR-1 recombinant protein In order to produce a human proto-oncogene HCCR-丨 specific antibody as a tumor marker substance for diagnosis of liver cancer, Some human proto-oncogenes (for the detection of GenBank & (10) i〇nn〇AF195651, jID.N〇s: 123_473, amino acid 4mDN〇s: 39i5 placed on pET-32(+)

The HCCR-lpET-32b (de) off (1) vector was prepared. The vector was transformed into the large intestine rod g el2i (atcc Accession Νο. 4) and induced by i to hit isopropyl called thiogalactosyl-purine (IPTG) (manufactured by Sigma Chemical Co.) to induce It was shown that 35 of the current HCCR1 fusion protein with 2〇Kda thioredoxin (Trix) tag protein fused thereto was produced and purified using 01/27149).

The His-Hind kit (manufactured by Novagen) (International Patent Publications) was used to immunofuse the purified recombinant egg to determine that it contained 15 1290226 Dali of a fusion protein of approximately 35 kDa (Fig. i). The purified recombinant protein of the ship is used as a fusion tumor cell line which can produce a monoclonal antibody for mouse immunization against the antigen-antibody binding reaction towel. <1·2> Immuneization of rabbit

A seven-month-old male New Zealand white rabbit was used as the HCH recombinant protein ultrafine can be prepared in the Example. The egg is mixed and emulsified in equal amounts from the igma chemical c〇 and F_d s. The sputum of each 〇 % ml will be inoculated with a straight chest. After the vaccination, the booster injection was performed at intervals of -week, and the antigen used for the immunization was emulsified using the incomplete adjuvant (manufactured by Sigma) and used in the same manner as the primary vaccination for u weeks. The emulsion was inoculated during this period. &lt;1_3&gt; Isolation of egg yolk resistance (IgY) A money sample was taken from the ear vein at intervals of -week and serum was isolated from the sample and used for various experimental measurements. Evaluation of antibody specificity in serum revealed that the antibody specifically reacts only with the human proto-oncogene HCCR-1 protein. &lt;Example 2&gt; Production of HCCR_1 monoclonal antibody&lt;2-1&gt; Immunization of mice In order to obtain an immunized mouse required for a fusion tumor cell line capable of producing HCOM monoclonal antibodies, the example is called 舛The prepared HCCW recombinant protein, Freund, s complete adjuvant (3), manufactured by Chemical Co.) and the F-face d, s incomplete side (manufactured by Gibb) were mixed. The resulting mixture was passed through the 岐_5_6 recording paper/e mice _, such as Lin, the mice were licked twice a week. At the time of the birth, the antibody titer of the mouse serum was measured by ELISA before the last enhancement of 16 1290226. Only the mouse material having an antibody titer that was 5 times larger than the negative control group was reinforced. Three days after the boost, cell fusion assays were performed. &lt;2-2&gt; Cell fusion (hybridoma) test Mouse plasma myeloma cells used as mother cells to be fused are obtained from the American Type of Culture Collection (ATCC), USA The SP_2 cell line was used for cell fusion only when the cells exhibited an exponential growth curve a week before the fusion assay. This cell line was grown in DMEM medium containing 10% fetal bovine serum. Three days after the last immunization, spleen cells were excised from Balb/c mice by a biopsy forceps and the spleen cells were placed in serum-free DMEM culture medium to prepare a spleen cell suspension. Then, the resulting solution was allowed to stand at room temperature to cause the connective tissue to settle down and then removed. The spleen cell suspension was centrifuged at 1, rpm, for 3 minutes, and then the spleen cells were exposed to Tris_NH4Cl solution containing Tris 20.6 g/L and NH4C1, 8.3 g/L to dissolve red blood cells, followed by passage through the culture solution. Centrifuge at 1,000 rpm for 3 minutes to wash 3 times, thereby bringing the number of cells to about 1 X 1 〇 8 cells/ml. In addition, 1.5 cubic centimeters of blood was collected from the iliac venous plexus of the mice as a positive control group in the subsequent experiments. SP-2 plasma myeloid cell tumor cells were mixed with spleen cells in a ratio of 1:1, and then fused using 5 〇% (w/v) polyethylene glycol 1500 (Boerhinger Mannheim Co., Germany). In order to screen only the fusion tumor cells, the cells produced by the cell fusion were suspended in a solution containing 15% fetal calf serum and containing 50 μ of hypoxanthine, 0.4 μ of aminopterin and 16 μΜ of thymidine solution. HAT solution in Waymouth broth and dispensed in 96-well microtiter plate (manufactured by Coming Co. USA) (100 μL/well). The fusion tumor cells were placed in a 5% CO 2 incubator at 37 ° C. Incubation under humidity conditions. 17 1290226 &lt;2-3&gt; Screening of a single tumor-resistant fusion cell line to screen for fusion tumor cells, screening for HCCR-1 by indirect enzyme-linked immunosorbent assay (ELISA) A cell in which a protein antigen specifically reacts. In other words, an absorbance is measured by an ELISA reader to screen a fusion tumor cell line which produces an antibody highly bindable to the purified HCCR-1 protein, and the fusion tumor cell is subjected to restriction dilution. Diluted to make the selected individual plants, thereby establishing a cell line composed of cells proliferating from the early cells. In order to select only the fusion tumor cell group prepared from the example &lt;2_2&gt; The fusion cell of the human pro-oncogene HCCR_1 recombinant protein antigen specifically reacted on the 10th day after cell fusion, and only the culture solution was taken out from the well with the proliferating cells and prepared for the preparation from the example ^-^ The HCCR-1 recombinant protein antigen isolated and purified by the E. coli transformant was subjected to ELISA. In detail, in order to suppress the non-specific immune response, a 96-well plate (Falc〇nc〇·, USA) was applied. % of the milk was removed from the PBS and allowed to stand at room temperature for a few hours, and the cultured Hcc cells (ATCC, USA) were added to each well in an amount of 2 X 1 〇 5 cells/well. After adding a microliter of the mixed tumor cell culture medium to a well, the antigen-antibody binding reaction was induced for 2 hours at 4 ° C, washed three times with PBS solution, and used as a secondary antibody, containing 2 % (w) diluted egg PBS buffer 1/1 〇, 000 over goat anti-rabbit (9) 〇c〇, de-made) reacted at * °C (100 μl / hole) for 1 hour. Place 100 μl via 10 ml of ai M disc buffer buffer-like acid tablet 5 〇), i mg 3.3,, 5.5,-tetrakilylidene aniline ( Tmb) (Sigma c〇_, said to be prepared) and 2 〇 microliters of Na hydrogen peroxide mixed with the solution was added to each hole to induce fermentin reaction. Maintain the enzyme reaction at room temperature for 15 minutes, then The same amount of 2 N acid-breaking solution was added to stop the enzyme reaction. The color of the silk was reduced. The single-body anti-system was continuously produced and produced, and the highly activated 18 1290226 fused cells were produced. The SHCCRd protein was produced. Among the fusion tumor cells with specific antibodies, only cells with antibody titer one-fold more than the negative control group in the ELISA assay were subjected to the step-by-step screening of the characteristics of the cells selected by the analysis. . After that, 'the first silk rides the HCCW New Zealand egg self-resistance with high (four) chicks combined with the activity of the fine antibody fusion tumor cells, and through repeated implementation of the phase _ procedures several times on the cell into the step, shame Cai Gaoling HCCW New Egg City Red and its specifically responded to the step-filtered fusion tumor cell group. In the medullary cell culture medium

At the time of the negative control group, _ showed cells that were 5 times larger than the yin-tested anti-determination hole. __ Cell_Bu 25 (Coming Co., USA) for proliferation. In this procedure, the antibody titer is measured more than 3 times to select the cells contained in the selection below the antibody titer that maintains the substantial amount, and some of the selected colonies are present in the _13〇C. under. In addition, in the antibody-producing fusion tumor cells, only three types of fusion tumor cells that are actively proliferating under high antibody titer are selected and maintained for proliferation by three colonizations, and are subsequently used for monoclonal antibodies. In production.

Fig. 2 shows the results of the application of HCCR-1 recombinant protein as an antigen for screening a fusion tumor cell line producing an HCCIM monoclonal antibody according to the present invention. k colony to limit the rare county 胄 (four). The number of aged tumor cells to be colonized was adjusted to include 230 fusion tumor cells in a ml of culture medium. Thereafter, 5 cells were dispensed into each of the 36 holes (U ml/hole) of the 96-well plate. The remaining culture solution (1 ml) was mixed with 4 ml of the culture solution, and the remaining 36 holes (〇) i cells were dispensed into the "ml/hole", and the last culture medium (1.4 ml) was mixed with the same amount of the culture medium, and 〇$ cells (〇ι ▲) were placed in the 24 holes respectively. After the day, when the selected plants were sufficiently grown, the presence of the produced antibodies was identified again from the culture solution, the liquid 19 1990226. The 6 positive plants were transferred to a 24-well plate and maintained, and finally the identification The presence of antibodies is generated. Then, only the positive colonies are transferred to a 25-ml vial and cultured for large-scale production. Some positive colonies are stored and maintained in liquid nitrogen tanks. 4&gt; Mass production of monoclonal antibodies In order to use the fusion tumor cell line established in the example &lt;2-2&gt;, a large amount of monoclonal antibodies against HccRq protein was produced, and the fusion tumor cells were intraperitoneally injected into Balb. About 0.5 days before /c mice, 0.5 ml of pristine (Sigma Co" USA Transplanted intraperitoneally into each mouse, thereby increasing the probability of producing intraperitoneal tumors. Subsequently, about 1×1〇7 fusion tumor cells are injected intraperitoneally to induce the production of ascites fluid, and Thereafter, the ascites fluid was collected from the abdominal hypertrophy of each mouse. The ascites fluid thus produced included the fusion tumor cells grown at a high concentration. Therefore, the collected ascites fluid was centrifuged at 10,000 rpm to precipitate the fusion tumor cells. Only the supernatant is recovered. Before the antibody protein is purified, the supernatant is stored in _7〇〇c. In order to purify the antibody from the ascites, protein-A/G agarose column chromatography is performed. (Pierce c〇., USA) and octanoic acid purification 'to isolate immunoglobulin (immun〇gi〇buiin). Finally, the concentration of immunoglobulin, that is, the concentration of immunoglobulin is via the use of Bio_Rad protein micro-assay set The group (made by Bi〇_Rad Lab·, USA) was measured by the Bradford method. The isotype of monoclonal antibody immunoglobulin

(Isotypes) were determined using a homotyped kit (Serotec Co., England) using a hemadsorbent reaction. As a result, the isotype isolated from the monoclonal antibody was identified as IgG2b 〇 &lt;Example 3&gt; by immunohistochemical staining in HCCR-1 monoclonal antibody to identify HC 20 1290226 c tissue proto-oncogene HCCR The specificity of -1 is such that in order to confirm according to the present invention, the monoclonal antibody against the human is used to control the Hcc tissue of the mosquito. Specifically, the HCCR_1 monoclonal antibody was treated with avidin-biotin/peroxidase complex (avidin_bi〇tin-P idase cqing (four) (ship) method ^ normal liver tissue and HCC _. After the money, I want to immunize _ chemically dyed fresh swollen weaving' and put ___ in liquid nitrogen to quickly knot, still underneath. Cut the tissue cut from HCC patients into 5 micron thickness, It was fixed in a propanil and placed in a 0.5% cerium peroxide/methanol solution for 1 minute to remove endogenous peroxidase activity, followed by washing with TBS. In addition, in order to press a specific immune response, the tissue was sliced. Exposure to normal horse serum (manufactured by Vector Lab., USA) for 3 minutes, and the diluted monoclonal antibody as the first antibody was added thereto at different concentrations, and then allowed to stand for 24 hours, followed by The obtained product was added with a biotinylated horse anti-rabbit IgG solution (manufactured by VectorLab., USA) as a second antibody, and allowed to stand for about 1 hour. The obtained product was further washed with TBS, and an anti-antigen was added thereto. Biotin-biotin-peroxidase complex (manufactured by Vector Lab., USA) as the first The antibody was allowed to stand for another 40 minutes, followed by thorough washing. The obtained product was exposed to a solution containing cerium peroxide and cerium (3,3·diaminobenzidine tetrahydrochloride) and observed under a microscope. The color appears. Then 'in view of the incidence of cells that can react with antibodies in HCC tissue samples, the disk distribution was counter-stained with hematoxylin and visualized with an optical microscope. As a result, it was confirmed that the HCC tissue contained a much larger amount of HCCR-丨 protein antigen in the liver cirrhosis 21 1290226 than the normal liver tissue and the prototype of HCC (Fig. 3). &lt;Example 4&gt; Using HCCR-1 alone Elisa by antibody The liver cancer was diagnosed by eusa using HCCR-1 monoclonal antibody as follows. First, the antibody was coated with the HCCr]-specific antibody isolated and purified from the fusion tumor cells. The second step was coated with The antibody on the plate is treated with a sample of the sample and reacted with it. In the third step, the HCCR-1 antigen-antibody binding in the sample is present. &lt;4-1&gt; HCCR-1 Coating a specific strain of HCCR-1 protein The body was placed in a % hole EUSA flat bottom plate, covered with a lid and allowed to stand at 16 ° C for 16 to 18 hours. The purified monoclonal antibody was in 〇·5 M carbonate buffer (pH 9). · 6) Dilute the concentration of 5 μg / liter and add 1 〇〇 microliter of diluted solution to each lake. For scaly enemies, the normal mouse blood will not be subtracted from f. Dilute 5 〇〇 times in the acid salt buffer and dispense into each well (1 μL/well). Then, the plate was drilled 4 times with a washing buffer, i.e., pBS (10) 7 4 containing 〇 5% Tween 2 。. Thereafter, in order to block the non-specific protein binding site, a PBS buffer solution containing 2% BSA blocking solution & Η 7·4) (3 μL/well) was dispensed into each well and allowed to The net was placed at 37 C for 2 hours. The blocking solution was then removed and vacuum packed for storage. &lt;4-2&gt; Reaction between the samples of the anti-cavity test specimens coated on the holes HCC and hepatitis liver serum, non-maternal and maternal serum and normal liver serum were used as samples. In order to obtain the fresh scale of the female side, the recombinant protein of HCCR] is labeled as G cfe microgram / 3⁄4 liter (ng / ), 2G ng / ml, 4 () ng / ml, (10) Prepare a standard antigen bath with nanograms/3⁄4 liters of '16 〇μg/ml, 32 〇 ng/ml diluted with _ nanograms/ml. Prepare a standard antigen bath VIII, 6'(]; '〇, £, 卩, and 0 22 1290226 An individual sample sample having a knives of 100 was assigned to each of the holes prepared with the HCCR-1 monoclonal antibody prepared in Example &lt;4-1&gt; and subjected to a reaction for 4 hours, followed by a reaction for 4 hours. Washed with washing buffer; strip 4 - human. Here, the fresh antigen prepared in the above is immersed in the yang (4) group and the normal mouse serum is used as the negative control group. &lt;4-3&gt; Antigen-antibody Combined detection of 100 μl of horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody 1 〇, 〇〇〇 _ dilution was added to each ―; and the plate was placed under 3rc Then, it was washed four times with the washing liquid. Then, 1 mg was dissolved as 33,5,5,4-tetramethylbenzidine (TMB) (manufactured by Sigma Co., USA). In 10 ml of citric acid The salt buffer was used to add a microliter of 35% per-emulsified hydrogen to prepare a substrate solution. The substrate solution prepared by microliter was dispensed to per-nano and reacted for 15 minutes at room temperature without violent light. Thereafter, (10) microliters of a 2 ΝΗΘ〇 4 solution was added to stop the reaction and the absorbance of 4 〇 5 nm was measured. For each sample, the absorbance of the HCCR_1 antigen was obtained by coating the antibody body of the individual antibody by the total absorbance of the hole coated with normal mouse serum as the positive control group and the hole coated with pBS as the negative control group. The remainder of the absorbance scale. In the way of _, the fresh (four) absorbance value is prepared. The average value of the fresh antigen solutions A and F is set as the cutoff value for later use.

The specimen is judged to be a positive or negative specimen. That is, when the absorbance of a sample is larger than the absorbance of the fresh antigen solution F, the hybrid is diluted and the absorbance is measured. A sample having an absorbance higher than the cutoff value is regarded as positive, and a sample having an absorbance lower than the cutoff value is identified as. Here, the average absorbance of the &amp; quasi-antigen bath A must be greater than or equal to the G and less than or equal to 0·2〇0. The average absorbance of the standard antigen solution F must be greater than or equal to i• and less than or equal to 3.000. 23 1290226 The cutoff value is reduced to ωμg/ml from the fresh scale of HCCR_1. Based on this cutoff value, the absorbance value of the sample is compared to be positive or negative by bullying. The comparison results of the liver cancer group, the hepatitis patient group, the maternal group and the normal group are shown in the figure. &lt;Example 5&gt; Determination of HCC diagnostic efficiency by ELISA using HCCR-1 monoclonal antibody In order to determine the diagnostic efficiency of liver cancer using the ELISA diagnostic kit and method of HCCR-1 monoclonal antibody, the HCCR-described in Example 4 was used. 1 Specific antibodies were subjected to ELISA measurements and measured using conventional alpha-fetoprotein (AFP) assays for comparison. The afp levels were measured using the ELSA2_AFP kit available from as Bio International, France. In order to determine whether the sample is positive or negative based on the diagnosis result, the cutoff value of AFP is set to 2 〇 nanogram/ml and the cutoff value of HCCR-1 is set to 1 〇 microgram/ml, which is obtained from the standard curve. The difference in reactivity between AFP positive and HCCR-1 positive in each HCC group, hepatitis group, and normal maternal group was compared with data obtained using the McNemar test. For the liver cancer group and the normal group, the HCCR-1 sensitivity, specificity, and pseudo-positive/negative ratio were measured in the 95% confidence interval. The liver cancer group was divided into two groups according to the tumor size of 2 cm. The difference in HCCR-1 positive reactivity between each individual group was compared to the data measured by the Fishery exact test (Fisherfs exact test). The difference between AFP positive reactivity and HCCR-1 positive reactivity for each group was compared to the data measured by the McNemar test. Use SAS variability of 6.12 in all statistical analyses and use a 0.05 level of significance (SAS/STAT Software: Changes and

Enhancements through Release 6· 12. Cary, NC: SAS Institute, 1997) o &lt;5-l&gt; AFP舆HCCR-1 kit for the correctness of the diagnosis of the HCC patient group Table 1 shows the HCCR-1 and AFP kits for Comparison of the diagnostic accuracy of 24 1290226 of the HCC group consisting of 97 liver cancer patients. These results show that the diagnostic accuracy according to Η·· is 94.9% 'which is significantly higher than the results obtained according to AFP, 7〇1% (chi-ΜΛ2=19 2, step P&lt;0 flat Bu McNemar test ). In particular, 27 of the 29 liver cancer patients (93.1%) were correctly diagnosed as Ηα:ΙΜ positive, and 3 of 68 liver cancer patients (4.4%) were diagnosed as AFP positive. , that is, diagnosed as HccRq negative. That is, the diagnosis is incorrect. Table 1 AF^ - positive negative total HCCR-1 positive 65 27 92 (94.9%) negative 3 - 2 5 ___ total 68 (70.1%) 29 197 (ϊδ〇%) 4-25 AFP and HCCR-1 kits for normal The diagnosis of the mother ship group is clearly evident from Table 2. In the normal mother group consisting of 72 women, the reactivity results of the two groups according to jjccn and AFP were positive. That is, 72 normal mother women are all positive, while only 11 maternal women (15.3%) are HCCR-1 positive, resulting in significant differences (cliiJV[A2=61, dfH, ρ&lt;〇·00( π, McNemar test). Therefore, it can be said that the diagnosis of AFP liver cancer is substantially meaningless to the normal maternal group. However, it can be determined that the diagnosis error rate can be significantly reduced according to the diagnosis of HCCR-1. Table 2 AFP positive negative total 25 1290226 HCCR-1 positive 11 0 11 (15.3%) negative 61 0 61 total 72 (100%) 0 72 (100%) &lt;5-3&gt; AFP and HCCR-1 kit for the diagnosis of hepatitis patients 3 shows the results of the AFP and HCCR-1 kits for the 72 patients with hepatitis. 2 out of 52 hepatitis patients (3.9%) were mistakenly diagnosed according to HCCR-1, and 52 Eleven of the patients with hepatitis (21.2%) were mistakenly diagnosed by the root-edge HCCR-1 (麁-MA2=7.3636, dfH, P=〇.〇〇67, McNemar test). Special, u One (90.9%) of the patients who were diagnosed as having liver cancer were diagnosed as positive. That is, the diagnosis of HCCR-1 is far more accurate than the diagnosis of AFP. Indeed. Table 3 AFP ------ positive negative total HCCR-1 positive 11 0 11 (15.3%) —— negative 61 0 6 Ϊ ·~- total 72 (100%) 0 72 (100%) ' &lt;5_4&gt The HCCR_1 kit as a liver cancer reading kit is affirmative as shown in Tables 4 and 5, 97 liver silkers and (2) normal people receiving money. The HCCM kit of the present invention has a sensitivity of 94 and its The % specificity, false positive and false negative were ^ and 4.4%, respectively. Moreover, the overall diagnostic accuracy was similar, and it was determined that the catering set of the present invention is very useful as a diagnostic tool. 26 1290226 Table 4

-------- Measurement —·~~~— ----— Value (%) —^ ～....................... ............. A 95% confidence interval sensitivity 92/97=94.9 ^^^_ 90·5 ~99.3 Specificity ~~~~~~~~ 109/121=90.1 — ^_ 84.8^95.4 False Positive S --- 12/104=11.5 ^^_ 5.4 ~17.6 False Negative -~~~--- 5/114=4.4 0.6 ~8.2 Positive Prediction Level -------- 92/104=88.5 82.3 ~94.6 Negative prediction level ~~~------- 109/114=95.6 91.8 ~99.4 Correctness-------- (92+109)/218=92.2 88.6 ~ 95.8 &lt;5_5&gt; Diagnosis of correctness of HCCR-1 kit according to 110^ tumor size~~-

Table 6 shows the test for the liver cancer patients with 85 tumors having a tumor of not less than 2 cm and the axis of the shaft having a shaft of not more than 2 cm. 珂 Having a size of not less than 2 centimeters _ size The patient has an AFP increase rate of about 72.9%, and (4) the patient has a positive rate of increase in the size of the patient who is not larger than the __ tumor size. It can be inferred that the tumor is a ruler and the foot is plus #^hccri 21 The base is . If it is on all swollen inches, it shows more than (four) sex. However, 27 1290226 HCCR-1 and AFP diagnostic kits did not show significant differences in tumor size. Among all tumor sizes, the HCCR-1 diagnostic kit showed significantly higher positive reactivity than the AFP kit (Ρ=〇·〇25, ρ=〇·〇〇ι). In tumor tissues of no more than 2 cm, HCCR_1 positive patients were 41.7% higher than AFP positive patients. In tumor tissues of not less than 2 cm, HCCR-1 positive patients were 22.4% higher than AFP positive patients. This means that the HCCR-1 diagnostic kit according to the present invention can be advantageously used to diagnose small-sized tumors. Table 6 Positive reactivity (%) Tumor size § AFP tlCCR-1 &lt;2 cm 6/12=50.0 11/12=91.7* 22 cm 62/85=72.9 81/85=95.31 — §Ρ&gt;0·05, The Dependent 2 assay and the Fisher, s exact test showed a positive reactivity difference between sputum and HCCR4 based on tumor size. *Ρ=0·025, according to the McNemar test, the positive reactivity difference between AFP and HCCR-1 according to tumor size of no more than 2 cm. 28 1 Ρ=0·001, according to the McNemar test, the positive reactivity difference between AFP and HCCR-1 according to the tumor size of not less than 2 cm. Industrial Applicability As described above, since the liver cancer reading kit containing the human proto-oncogene HCCRq-specific antibody according to the present invention is an immunodiagnostic jade using patient serum, it has a higher quality than the conventional marriage test tool. And re-dissociation, so it can be advantageously used for the early diagnosis of Lai and the diagnosis of small HCC.

Claims (1)

1290226 5. The kit according to item 1 of the patent application, wherein the labeling substance of the secondary antibody composition is selected from the group consisting of horseradish peroxidase (HRP) alkaline phosphatase, colloidal gold, luciferin, and Among the groups of dyes.